Smooth muscle α 1D ‐adrenoceptors mediate phenylephrine‐induced vasoconstriction and increases in endothelial cell Ca 2+ in hamster cremaster arterioles

Background and purpose: α 1 ‐Adrenoceptor agonists induce Ca 2+ ‐transients in endothelial cells (ECs) of arterioles. However, the presence of α 1 ‐adrenoceptors on arteriolar ECs has not been excluded, and the identity of α 1 ‐adrenoceptor subtypes in arterioles only has been inferred from pharmacology. Therefore, we determined which subtypes were expressed by vascular smooth muscle cells (VSMCs) and ECs, and which subtype mediated α 1 ‐adrenoceptor‐induced constriction. Experimental approach: EC Ca 2+ ‐transients in isolated, cannulated hamster cremasteric arterioles or freshly isolated ECs were studied using Fura 2. Arteriolar diameter was measured by video microscopy. α 1 ‐Adrenoceptor expression was assessed by western blot of whole‐arteriolar homogenates and real‐time RT‐PCR on enzymatically isolated VSMCs and ECs. Key results: Phenylephrine‐induced constriction and EC Ca 2+ ‐transients were abolished by the α 1 ‐adrenoceptor antagonist prazosin (30 n M ) in arterioles. Phenylephrine‐induced constriction was inhibited by the α 1D ‐adrenoceptor antagonist BMY 7378 ( K B =2.96 n M ) and the α 1A ‐adrenoceptor antagonist 5‐methylurapidil ( K B =4.08 n M ), suggesting a significant role for α 1D ‐adrenoceptors. Western blots confirmed α 1D ‐adrenoceptor expression, but did not detect α 1A ‐adrenoceptors. VSMCs expressed α 1D ‐ and α 1A ‐, but not α 1B ‐, adrenoceptor transcripts. No α 1 ‐adrenoceptor transcripts were detected in ECs. Neither phenylephrine (10 μ M ) nor noradrenaline (0.1–1 μ M ) elicited Ca 2+ ‐transients in freshly isolated ECs, whereas the endothelium‐dependent vasodilators methacholine (1 μ M ) and substance P (100 n M ) consistently increased Ca 2+ . Conclusions and implications: We reject the hypothesis that hamster cremasteric arteriolar ECs express α 1 ‐adrenoceptors and conclude that α 1 ‐adrenoceptor agonists predominantly act on VSMC α 1D ‐adrenoceptors to cause vasoconstriction and a subsequent rise in EC Ca 2+ . British Journal of Pharmacology (2008) 155 , 514–524; doi: 10.1038/bjp.2008.276 ; published online 7 July 2008 www.bjcancer.com